Tuberculosis is one of the most important world health problems, responsible for 450,000 deaths in 2012. Increases in the occurrence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) have become an increasingly problematic health crisis. Clearly there is a critical unmet need for the development of novel antibiotics against M. tuberculosis (MTB) to overcome resistance to current therapeutics. However, only one new drug (bedaquiline) with a novel target, the mycobacterial ATP synthase, has been approved for the treatment of tuberculosis in the last 40 years. The lack of success in inhibiting novel targets suggests that the reinvestigation of targets of previously effective drugs may be a better approach. The MTB RNA polymerase (RNAP) is a proven and attractive target because it is essential for bacterial survival, and there is low similarity between prokaryotic and eukaryotic RNAPs. The rifamycins (RIFs) are very potent inhibitors of MTB RNAP; however, these agents suffer from resistance (RIFR) via mutation of the target RNAP and drug-drug interactions that result from RIF activation of the human pregnane X receptor (hPXR) (particularly problematic in TB/HIV co-infection). We propose a multi-disciplinary and comprehensive program that involves structure-based analogue synthesis, high-throughput screening (HTS), in vitro RNAP inhibition evaluation, X-ray crystal structure determinations of inhibitor*RNAP complexes, and studies to minimize hPXR activation towards uncovering novel agents to address current MTB treatment limitations. We have assembled a team with skills and experience in biochemistry and enzymology of RNA and bacterial RNA polymerase, decades of synthetic medicinal chemistry and molecular modeling in the pharmaceutical industry, groundbreaking structural biology of bacterial RNA polymerase, and internationally-recognized expertise in the microbiology of M. tuberculosis. The recent publications (four papers, one review, and one patent application) of our team demonstrate the proof of principle of our approach. Our very first set of RIF analogues exhibit enhanced activity against RIFR RNAP, in one case a reduction in hPXR activation, and bind to the RNAP in the designed mode as shown by our X-ray crystal structures. These X-ray crystal structures now provide a conceptual framework for our development of improved RIFs. Recent studies by Ebright and co-workers (Zhang et al., (2014) eLife 3 e02450, 3994528) provide further proof of principle that elaboration of the rifamycin core can yield enhanced activity against RIFR MTB. We have also developed an efficient in vitro RNAP assay, scalable for HTS, and will use this as a complementary approach for discovering novel RNAP inhibitors. It is our expectation that the fully articulated campaign described in this application will yield novel candidates for drug development and ultimately improve treatment for tuberculosis, especially in patients with HIV-TB co-infection and drug-resistant tuberculosis.